Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

Abstract

We have investigated factors that influence the production of native-like soluble, recombinant trimers based on the env genes of two isolates of human immunodeficiency virus type 1 (HIV-1), specifically 92UG037.8 (clade A) and CZA97.012 (clade C). When the recombinant trimers based on the env genes of isolates 92UG037.8 and CZA97.012 were made according to the SOSIP.664 design and purified by affinity chromatography using broadly neutralizing antibodies (bNAbs) against quaternary epitopes (PGT145 and PGT151, respectively), the resulting trimers are highly stable and they are fully native-like when visualized by negative-stain electron microscopy. They also have a native-like (i.e., abundant) oligomannose glycan composition and display multiple bNAb epitopes while occluding those for nonneutralizing antibodies. In contrast, uncleaved, histidine-tagged Foldon (Fd) domain-containing gp140 proteins (gp140 UNC -Fd-His), based on the same env genes, very rarely form native-like trimers, a finding that is consistent with their antigenic and biophysical properties and glycan composition. The addition of a 20-residue flexible linker (FL20) between the gp120 and gp41 ectodomain (gp41 ECTO ) subunits to make the uncleaved 92UG037.8 gp140-FL20 construct is not sufficient to create a native-like trimer, but a small percentage of native-like trimers were produced when an I559P substitution in gp41 ECTO was also present. The further addition of a disulfide bond (SOS) to link the gp120 and gp41 subunits in the uncleaved gp140-FL20-SOSIP protein increases native-like trimer formation to ϳ20 to 30%. Analysis of the disulfide bond content shows that misfolded gp120 subunits are abundant in uncleaved CZA97.012 gp140 UNC -Fd-His proteins but very rare in native-like trimer populations. The design and stabilization method and the purification strategy are, therefore, all important influences on the quality of trimeric Env proteins and hence their suitability as vaccine components. IMPORTANCESoluble, recombinant multimeric proteins based on the HIV-1 env gene are current candidate immunogens for vaccine trials in humans. These proteins are generally designed to mimic the native trimeric envelope glycoprotein (Env) that is the target of virus-neutralizing antibodies on the surfaces of virions. The underlying hypothesis is that an Env-mimetic protein may be able to induce antibodies that can neutralize the virus broadly and potently enough for a vaccine to be protective. Multiple different designs for Env-mimetic trimers have been put forth. Here, we used the CZA97.012 and 92UG037.8 env genes to compare some of these designs and determine which ones best mimic virus-associated Env trimers. We conclude that the most widely used versions of CZA97.012 and 92UG037.8 oligomeric Env proteins do not resemble the trimeric Env glycoprotein on HIV-1 viruses, which has implications for the design and interpretation of ongoing or proposed clinical trials of these proteins. R ecombinant, soluble envelope glycoprotein (Env) gp140 trimers from...

Glycosylation Benchmark Profile for HIV-1 Envelope Glycoprotein Production Based on Eleven Env Trimers

“…Compared to these irregularly folded proteins, the PGT151-purified C97ZA.012 trimers are enriched for high-mannose glycans (56). Therefore, in some cases the elimination of inappropriately assembled proteoforms with concomitantly aberrantly processed glycans is achievable using a bNAb column, as has been described previously elsewhere (56).…”

“…This purification method was necessary to isolate appropriately folded C97ZA.012 trimers because a substantial subset of the nonselected gp140 proteins adopts multiple nonnative conformations (56). Compared to these irregularly folded proteins, the PGT151-purified C97ZA.012 trimers are enriched for high-mannose glycans (56). Therefore, in some cases the elimination of inappropriately assembled proteoforms with concomitantly aberrantly processed glycans is achievable using a bNAb column, as has been described previously elsewhere (56).…”

“…PGT151 is a conformationally selective antibody, and a glycan is part of its epitope. This purification method was necessary to isolate appropriately folded C97ZA.012 trimers because a substantial subset of the nonselected gp140 proteins adopts multiple nonnative conformations (56). Compared to these irregularly folded proteins, the PGT151-purified C97ZA.012 trimers are enriched for high-mannose glycans (56).…”

“…The protein was purified first by using a 2G12-affinity column and then by SEC (59,61,62). The C97ZA.012 SOSIP.664 trimer and the C97ZA.012 gp140(-)FT were expressed in 293F cells by transient transfection, as described previously (56). The uncleaved C97ZA.012 gp140(-)FT, which contains a C-terminal His tag and a trimeric fibritin foldon at its C terminus, was first purified by Ni-NTA affinity chromatography, followed by SEC (56).…”

“…The C97ZA.012 SOSIP.664 trimer and the C97ZA.012 gp140(-)FT were expressed in 293F cells by transient transfection, as described previously (56). The uncleaved C97ZA.012 gp140(-)FT, which contains a C-terminal His tag and a trimeric fibritin foldon at its C terminus, was first purified by Ni-NTA affinity chromatography, followed by SEC (56). The C97ZA.012 SOSIP.664 trimer was first purified via the use of a PGT151 bNAb affinity column and then by SEC (56).…”